Calvin M C, Blouquit Y, Garel M C, Prehu M O, Cohen-Solal M, Rosa J, Rosa R
INSERM U91, CNRS UA 607 Hôpital Henri Mondor, Créteil, France.
Biochimie. 1990 May;72(5):337-43. doi: 10.1016/0300-9084(90)90029-g.
Bisphosphoglycerate mutase (EC 5.4.2.4.) is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate (glycerate-2,3-P2) an effector of the delivery of O2 in the tissues. In addition to its main synthase activity the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. Using a prokaryotic expression system, we have developed a recombinant system producing human bisphosphoglycerate mutase in E coli. The expressed enzyme has been extracted and purified to homogeneity by 2 chromatographic steps. Purity of this enzyme was checked with sodium dodecyl sulfate polyacrylamide gel and Cellogel electrophoresis and structural studies. The bisphosphoglycerate mutase expressed in E coli was found to be very similar to that of human erythrocytes and showed identical trifunctionality, thermostability, immunological and kinetics' properties. However, the absence of a blocking agent on the N-terminus results in a slight difference of the electrophoretic mobility of the enzyme expressed in E coli compared to that of the erythrocyte.
二磷酸甘油酸变位酶（EC 5.4.2.4.）是一种红细胞特异性酶，其主要功能是合成2,3-二磷酸甘油酸（甘油酸-2,3-P2），这是一种组织中氧气输送的效应物。除了其主要的合酶活性外，该酶还表现出磷酸酶和变位酶活性，两者在反应中均涉及2,3-二磷酸甘油酸。我们使用原核表达系统开发了一种在大肠杆菌中产生人二磷酸甘油酸变位酶的重组系统。通过两步色谱法提取并纯化表达的酶至同质状态。用十二烷基硫酸钠聚丙烯酰胺凝胶和Cellogel电泳及结构研究检查该酶的纯度。发现大肠杆菌中表达的二磷酸甘油酸变位酶与人红细胞中的非常相似，并表现出相同的三功能、热稳定性、免疫学和动力学特性。然而，与红细胞相比，大肠杆菌中表达的酶N端缺少封闭剂导致其电泳迁移率略有差异。
